Shigella spp. continue to be a leading cause of dysentery and diarrhea annually worldwide. Shigella is unique among Gram-negative enteric pathogens in that it accesses the cytoplasm of host cells and assembles actin into long tails, which propel bacterial spread through tissues. The investigators have previously shown that the Shigella outer membrane protein IcsA is sufficient for actin assembly and that IcsA is localized to a single pole of the bacillus. The molecular mechanisms involved in the unipolar localization of IcsA are unknown. Their data demonstrate that IcsA is directly targeted to the bacterial pole. In conjunction with this, they have developed constructs that provide the tools necessary to directly address the molecular mechanisms of unipolar targeting of IcsA, a major goal of this proposal. In contrast, IcsA that is uniformly distributed over the surface of certain E. coli strains is able to mediate actin tail assembly without capping of the IcsA. This proposal also specifically addresses the function of unipolar localization of IcsA in Shigella pathogenesis, which they are now in a position to test critically. The Specific Aims of this proposal are: (1) the identification and characterization of residues and domains of IcsA required for its unipolar localization; (2) the identification of Shigella proteins that directly interact with IcsA and analysis of their potential role in IcsA unipolar localization; and (3) an assessment of the role of IcsA unipolarity in Shigella pathogenesis. This application proposes to obtain information that will provide insight into the molecular mechanisms of unipolar targeting of the S. flexneri virulence factor IcsA. Further, their studies will elucidate the role of IcsA unipolarity in the pathogenesis and virulence of Shigella. Finally, their studies will likely provide insight into the fundamental mechanisms that mediate three-dimensional targeting of proteins in bacteria and the molecular characteristics of the bacterial old pole that distinguish it from the new pole and the sides of the bacillus.